Method for the preselection of drugs for protein misfolding diseases

Abstract

The invention provides a method that gives direct information about the intervention of a potential drug on the secondary structure distribution of a targetbiomolecule, i.e., for a disease with misfolded protein, such as neurodegenerative diseases in a complex body fluid. The secondary structural change is monitored by vibrational spectroscopy. The method can be applied for prescreening of drug candidates for targeting of specific biomolecules. The effect of the drug on the secondary structure distribution is monitored label-free in real time and provides thereby direct information about the efficacy of the potential drug.

The invention claimed is: 
     
       1. A drug-screening assay comprising the steps:
 contacting a flux of a sample of a complex body fluid comprising a target protein, wherein the target protein undergoes secondary structure changes associated with a disease, with an infrared (IR) cell comprising an infrared sensor element comprising:
 an internal reflection element comprising a core of an infrared transparent material and at least one antibody capable of specific and conformationally independent binding to the target protein, wherein the antibody is covalently attached to at least one surface of said internal reflection element; wherein said contacting loads said at least one antibody with the target protein; 
 submitting an IR beam through said IR cell; and obtaining a first infrared spectrum therefrom; 
 contacting the IR cell with a solution comprising a potential drug; 
 submitting an IR beam through said IR cell; and obtaining a second infrared spectrum therefrom, and subtracting a reference spectrum of the potential drug from the second infrared spectrum; and 
 analyzing: i) the first infrared spectrum and ii) the second infrared spectrum after subtraction of the reference spectrum of the potential drug, to evaluate the effect of the potential drug by determining the secondary structure distribution of the target protein in the sample before and after application of the potential drug, wherein an upshift or disappearance of an amide I band characteristic for β-sheets in the second infrared spectrum after subtraction of the reference spectrum of the potential drug relative to the corresponding amide I band in the first spectrum is indicative of the efficacy of the potential drug; wherein the potential drug is an antibody or comprises amide bonds. 
 
 
     
     
       2. The method of  claim 1 , wherein
 (i) the infrared transparent material of the IR cell is independently selected from gallium arsenide, silicon, germanium, zinc, selenide and diamond and/or 
 (ii) the target protein is an amyloidogenic peptide or a (poly-) peptide of health-status dependent, characteristic secondary structure composition; and/or 
 (iii) the sample of the complex body fluid with Tau target protein is human cerebrospinal fluid (CSF), serum or blood plasma. 
 
     
     
       3. The method of  claim 1 , wherein said infrared sensor element comprises
 a germanium internal reflection element being of trapezoid or parallelogram shape and being transparent in the infrared with sufficient signal to noise ratio to detect the amide I band, and 
 wherein the at least one antibody is and being directly grafted covalently attached to at least one surface of said internal germanium reflection element by a method comprising:
 silanization with short silane linkers or by thiolation with short thiol linkers, 
 reacting freely accessible amine groups of said at least one antibody with amine-reactive groups on the short silane/thiol linkers, and 
 blocking remaining amine-reactive groups on the short silane/thiol linkers with a blocking substance not cross-reacting with the biomarker protein. 
 
 
     
     
       4. The method of  claim 1 , wherein the internal reflection element
 (i) is a germanium monocrystal; and/or 
 (ii) allows for or provides for more than one passages of the infrared light through the reflection element; and/or 
 (iii) is further suitable for the parallel analysis by another optical method including detection of fluorescence at different wavelengths; and/or 
 (iv) the blocking substance not cross-reacting with the target protein is selected from casein, ethanolamine, L-lysine, polyethylene glycols, albumins and derivatives thereof. 
 
     
     
       5. The method of  claim 3 , wherein the silane and thiol linkers comprise homogenous silane and thiol linkers, mixtures of silane linkers and mixtures of thiol linkers, and have an effective linker chain length (combined number carbon and heteroatoms) of not more than 20 atoms or not more than 15 atoms. 
     
     
       6. The method of  claim 1 , wherein the target protein is a Tau protein and wherein a shift of the amide I band to any value indicative for the Tau protein secondary structure is indicative for the efficacy of the potential drug. 
     
     
       7. The method of  claim 1 , wherein the potential drug is for the treatment of a neurodegenerative disease. 
     
     
       8. The method of  claim 2 , wherein the infrared transparent material is germanium. 
     
     
       9. The method of  claim 4 , wherein the germanium monocrystal is a trapezoid cut germanium monocrystal. 
     
     
       10. The method of  claim 5 , wherein the silane linkers have one of the following formulas:
   X 3 Si—(CH 2 ) n —Y—(CH 2 ) n′ —Z,  (i)
 
   X 2 R 1 Si—(CH 2 ) n —Y—(CH 2 ) n′ —Z or  (ii)
 
   X(R 1 ) 2 Si—(CH 2 ) n —Y—(CH 2 ) n′ —Z,  (iii)
 
 
       and the thiol linkers have the following formula:
   WS—(CH 2 ) n -Y—(CH 2 ) n ′-Z,  (iv)
 
 
       wherein W is H or R 1 S—, X at each occurrence is independently selected from halogen and C 1-6  alkoxy, n is an integers of 1 to 10, n′ is an integer of 1 to 5; R 1  at each occurrence is independently selected from C 1-6  alkyl, Y is selected from a chemical bond, O—, —CO—, —SO 2 —, —NR 2 —, —S—, —SS—, —NR 2 CO—, —CONR 2 —, —NR 2 SO 2 — and —SO 2 NR 2 — (wherein R 2  is H or C 1-6  alkyl), and Z is an amine-reactive group including —CO 2 H, —SO 3 H and ester derivatives thereof. 
     
     
       11. The method of  claim 1 , wherein the potential drug is an antibody. 
     
     
       12. The method of  claim 6 , wherein the shift of the amide I band is a shift to the amide 1 band maximum. 
     
     
       13. The method of  claim 6 , wherein the shift for a fibrillary fraction of the Tau protein from of 1626 cm −1  to 1655 cm −1  is indicative of the efficacy of the potential drug. 
     
     
       14. The method of  claim 6 , wherein the shift for the total fraction of the Tau protein from 1636 cm −1  to 1655 cm −1  is indicative of the efficacy of the potential drug. 
     
     
       15. The method of  claim 10 , wherein X is independently selected from methoxy and ethoxy groups. 
     
     
       16. The method of  claim 10 , wherein n is 3 and n′ is 2. 
     
     
       17. The method of  claim 10 , wherein n is 8 and n′ is 4. 
     
     
       18. The method of  claim 1 , wherein
 (i) the target protein is an Aβ peptide, or 
 (ii) the target protein is a Tau protein. 
 
     
     
       19. The method of  claim 1 , wherein the target protein is an Aβ peptide and a shift of the amide I band to any value indicative for the Aβ peptide secondary structure is indicative of the efficacy of the potential drug. 
     
     
       20. The method of  claim 10 , wherein the infrared sensor element is obtainable by:
 (A) silanization and in the linkers of formulas (i) to (iii), wherein X is independently selected from C 1-6  alkoxy-groups, Y is —NHCO—, Z is —CO 2 H or an ester derivative thereof, and n is an integer of 1 to 5 and n′ is an integer of 1 to 3; or 
 (B) thiolation and in the linkers of formula (iv), wherein W is H, Y is a chemical bond, Z is —CO 2 H or an ester derivative thereof, and n is an integer of 1 to 8 and n′ is an integer of 1 to 5. 
 
     
     
       21. The method of  claim 20 , wherein X is independently selected from methoxy and ethoxy groups. 
     
     
       22. The method of  claim 20 , wherein n is 3 and n′ is 2. 
     
     
       23. The method of  claim 20 , wherein n is 8 and n′ is 4.